High-risk human papilloma virus (hereinafter, “HPV”) types and 18 are major factors of cervical cancer and cervical dysplasia, and become causes of other genital cancers and a head and neck squamous cancer. Cervical cancer is one of the most general types of malignant tumors of women. Although the incidence of invasive cervical cancer has been slowly reduced, the invasive cervical cancer is a still most frequent cancer of women in developing countries, which holds 25% of female cancers. HPV is a small DNA virus having approximately 8,000 base sequences which causes benign or malignant tumors. So far, depending on a genomic difference, at least about 100 HPV subtypes have been identified, and genotypes of approximately HPVs have been completely analyzed. Among these types, high-risk HPV types (e.g., HPV-16, 18, 31, 33, 35, 45, 51, 52, and 56) relate to almost 90% of cervical cancer. At least 50% of cervical cancer infected with HPV relate to HPV type 16, and followed by HPV type 18 (12%), HPV type 45 (8%), and HPV type (5%). These HPVs encode 2 oncogenic proteins, which are, protein E6 and E7. Both proteins are involved in HPV-mediated cell immortalization and cell transformation. The oncogenic E6 protein binds to wild-type p53 tumor suppressor protein to thereby degrade p53 through an ubiquitin pathway. On the other hand, the E7 protein directly binds to Rb to thereby overphosphorylate Rb. At first, E6 forms a complex with an E6-associated protein (E6-AP) which is an E3 ubiquitin-protein ligase. Then, the E6/E6-AP complex binds to and ubiqutinate wild-type p53, and then interferes with p53-mediated cellular reaction to DNA damage. Mostly, the p53 tumor suppressor protein is regulated by Mdm2-mediated ubiquitination, however, in HPV-infected cervical cancer cells, degradation of p53 is completely changed to E6-mediated ubiquitination from Mdm2-mediated ubiquitination. Thus, unlike many other cancers, most cases of HPV-infected cervical cancer have the wild-type p53 gene. However, an expression level of the p53 protein is very low due to the consistent degradation by the E6 protein. Particularly, the HPV E6 protein has been significantly noticeable as a specific target for killing just cervical cancer cells. These strategies, targeting to E6 or the E6/E6-AP complex, include various treatment.
Examples include: use of a cellular toxin agent, an inhibitor to release zinc of the E6 oncogenic protein, an epitope peptide (mimotope) mimicking E6-AP, anti-E6 ribozyme, a peptide aptamer which is targeted to the E6 oncogenic protein of a virus, siRNA which is targeted to the E6 oncogenic protein of the virus, and a combined treatment thereof. Recently, it has been proven that siRNA selectively silences an intrinsic gene in animal cells, and as well as, selectively silences a viral gene in a disease caused by a virus. RNA interference (RNAi) due to transfection of siRNA has been emerged as a novel therapy for treating viral infection of the human. siRNA, which is targeted to E6 and E7 genes in HPV-infected cervical cancer cells, causes p53 and pRb accumulation which leads to apoptosis or cell senescence. For an HPV-16 infected cervical cancer cell line and an HPV-18 infected cell line, it has been found that RNAi, which is targeted to E6 and E7 oncogenes of viruses, selectively sciences expression of these proteins.
Meanwhile, efficacy of a nucleic acid having various modifications for nucleic acids (for example, in a base, a sugar and/or phosphate) is enhanced by inhibiting degradation caused by serum ribonuclease. Several examples describing sugar, base and phosphate modifications, which may be introduced to a nucleic acid, are known in the art. For example, an oligonucleotide is modified to enhance stability and/or enhance the biological activity through a modification by a nuclease-resistant group, for example, through 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, and 2′-H nucleotide base modification (see Eckstein et al., PCT Laid-open Publication WO 92/07065; document [Perrault et al., Nature 344:565-568, 1990]; document [Pieken et al., Science 253: 314-317, 1991]; document [Usman and Cedergren, Trends in Biochem. Sci. 17: 334-339, 1992]; Usman et al. PCT Laid-open Publication WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and document [Beigelman et al., J. Biol. Chem., 270:25702, 1995]; Beigelman et al., PCT Laid-open Publication WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., PCT Laid-open Publication WO 98/13526; Thompson et al., U.S. Patent Application No. 60/082,404 (filed on Apr. 20, 1998); document [Karpeisky et al., Tetrahedron Lett., 39:1131, 1998]; document [Earnshaw and Gait, Biopolymers (Nucleic acid Sciences) 48:39-55, 1998]; document [Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998]; and document [Burlina et al., Bioorg. Med. Chem. 5: 1999-2010, 1997]). Similar modifications may be used to modify the nucleic acid of the present invention.
In 1999, as a result of combined therapy of cisplatin-based chemotherapy and radiotherapy, survival rate of women who have a severe cervical cancer in local has been significantly improved. Currently, cisplatin is a DNA-damaging drug which is widely used to treat cancers including ovarian, cervical, head and neck, non-small cell lung cancers and so forth. More recently, a working mechanism of a medicine based on platinum has been investigated. However, it is still not fully understood about a process in cells including DNA repair, cell death, cell cycle trajectory, signaling of DNA damage, and regulation in absorption and secretion of a drug due to cisplatin treatment. In HPV-18 HeLa cells, the p53 protein is escaped from E6-mediated degradation and preferentially accumulated in nucleolus of a nucleus after cisplatin treatment. Also, HPV-16 SiHa cells recover p53 function by simultaneous radiotherapy and cisplatin treatment, thereby increasing radiosusceptibility.
Therefore, as a result of attempting to investigate an effective siRNA having a novel sequence by imparting a chemical modification to E6/E7-specific siRNA so that the siRNA may show the anti-cancer effect, alone or in a complex combination, or show a synergistic effect when performing combination therapy with conventional chemotherapy or radiotherapy, the present inventors have found that nucleotides listed on following Examples and Claims and particular combinations thereof reduce expression of relating proteins TP53 and E7, and HPV E6 mRNA, and induce cell death, and also experimentally proven that efficacy achieved when used alone or in combination with anti-cancer agents is much better than that of RNA, which does not have a base sequence residue modification.
Throughout the specification, numerous journals and patent documents are referenced, and the citation is indicated. Disclosures of cited journals and patent documents are incorporated herein in their entireties by reference to more clearly describe the level of the technical field to which the present invention belongs and features of the present invention.